Although reports on microsurgical epididymal sperm aspiration with IVF appeared in the literature as early as 1984, the first live birth was not reported until 1990. As it was first described, the procedure of microsurgical epididymal sperm aspiration involved careful dissection of the epididymis under the operating microscope and incision of a single tubule.
Once incised, fluid spills from the epididymal tubule and pools in the epididymal bed. This pooled fluid is then aspirated. Because the epididymis is richly vascularized, this technique invariably leads to contamination by blood cells that may affect sperm fertilizing capacity in vitro.
Our initial experience with this "pool and aspirate" technique was unsuccessful due to contamination of the aspirated sperm with blood products. This appeared to result in impaired sperm function and the inability to fertilize oocytes in vitro. For this reason we developed a technique of micropuncture of the epididymal tubule to avoid blood product contamination. This technique, combined with improved ovarian stimulation techniques and micromanipulation of retrieved sperm, has resulted in markedly improved fertilization and pregnancy rates in our patients with unreconstructable reproductive tract obstruction. The technique of microsurgical epididymal sperm retrieval offers the advantages of minimizing contamination of epididymal fluid with blood cells, repeated aspirations can be performed, and aspiration of sufficient quantities of fluid for immediate use as well as for cryopreservation are possible.
(1): Aspiration Device
In order to aspirate fluid from within the epididymal tubules a device is needed that is sharp and fine enough to be able to pierce the tubule successfully, and avoid the extensive, delicate network of Vessels that cover the epididymis. To achieve these goals micropipettes with tip widths of 250 to 350 um were hand drawn from glass tubing with an outer diameter of 0.9 mm and inner diameter of 0.6 mm, and then hand sharpened on a grinding wheel. The micropipette is then attached to silicone tubing and a three-way stopcock. Two syringes are attached to the stopcock, a 1 cc tuberculin syringe and a 10 cc glass syringe. The tuberculin syringe collects the epididymal fluid when sufficient fluid is obtained, while the glass syringe provides fine control of the aspiration as well as rapid equilibration of pressure to avoid aspiration of blood outside of the epididymal tubules (Figure 2).
A unique micropuncture pipet holding apparatus, MESA- Holder has been developed and patented (Schlegel, Li and & Goldstein) at Cornell (Figure 3). Its unique 180° angle adjustable pipet holding system simplifies the procedure of micropuncture epididymal sperm retrieval. The micropuncture technique is an atraumatic technique that limits damage to the epididymal tubules and avoids potential blood cell contamination of the epididymal fluid, while yielding high quantities of motile spermatozoa. In this system it is imperative that the epididymal fluid never contact the glass syringe as the sperm may adhere to the glass surface.
(2): Operative Procedure
The patient is explored through a midline scrotal incision. The testis is delivered and tunica vaginalis is opened to expose the epididymis (Figure 4).
The operating microscope is brought into the sterile field and the epididymis is examined under 8 to 15X magnification. The obstructed epididymis has a characteristic appearance. The tail of the epididymis has dilated yellow tubules due to the predominance of macrophages and degenerating sperm. The first puncture for aspiration is made proximal to these yellow tubules. If the tubules can be clearly visualized with the epididymal tunic intact, the puncture can be made through the tunic (Figure 5).
If however the tubules are obscured by the tunic, a linear opening in the tunic is made with a 15 degree microknife after coagulating the surface with the bipolar electrocautery. An alternative approach is to incise tubules and gather fluid after it flows out of the tubules.
With the assistant stabilizing the testicle, a suitable tubule is punctured by the operating surgeon and fluid is gently aspirated (Figure 6). The assistant can facilitate retrieval by gently compressing the testis and epididymis. When there is no longer flow, the retrieved fluid is back flushed through the system using 0.5 cc of human tubal fluid and handed to the in vitro fertilization team who are standing by in the operating room.
They examine the fluid immediately under the microscope to assess sperm count and motility. Sequential micropunctures can be performed until optimal sperm quality has been obtained. Typically approximately 100 x 106 sperm with good motility are retrieved using this MESA approach. Because sperm in the epididymal fluid are highly concentrated (roughly 1 x 106 /ul), only microliters quantities of epididymal fluid are needed to be retrieved. In this way, MESA provides for more than adequate numbers of sperm for immediate use with ICSI, as well as for sperm cryopreservation. (Figure 7)
If optimal sperm quality has not been found in the caput or body of the epididymis, fluid can be retrieved from the efferent ductules with the micropuncture technique. The efferent ductules arise from the superior pole of the testis, just superior to the testicular vessels. Therefore, with the assistant orienting the testicle properly, the operating surgeon can expose the efferent ductules by incising the tunic at the junction of the epididymis and testis. In this way the caput epididymis is dissected bluntly off of the testis. After adequate fluid has been retrieved the puncture sites are closed with 10-0 monofilament nylon sutures. The tunic incisions are closed with 6-0 polypropylene or larger. The tunica vaginalis is closed in a water tight fashion to avoid inflammation and adhesions that could complicate future explorations. Epididymal fluid samples are taken from the operating room to the in vitro fertilization laboratory where they are subjected to mini-Percoll discontinuous gradient centrifugation, swim-up, and/or sedimentation to remove debris, macrophages and blood products.